PUBLICATION
Identification and Characterization of a Selective Allosteric Antagonist of Human P2X4 Receptor Channels
- Authors
- Ase, A.R., Honson, N.S., Zaghdane, H., Pfeifer, T.A., Seguela, P.
- ID
- ZDB-PUB-150120-2
- Date
- 2015
- Source
- Molecular pharmacology 87(4): 606-16 (Journal)
- Registered Authors
- Keywords
- Calcium, Imaging with fluorescent indicators (e.g. Ca2+ imaging), Microglia, Neuroinflammation, Patch clamp methods, Purinergic
- MeSH Terms
-
- Allosteric Regulation
- Aminopyridines/chemistry
- Aminopyridines/pharmacology*
- Animals
- Calcium/metabolism
- Databases, Chemical
- HEK293 Cells
- Humans
- Mice
- Patch-Clamp Techniques
- Phenylurea Compounds/chemistry
- Phenylurea Compounds/pharmacology*
- Purinergic P2X Receptor Antagonists/chemistry
- Purinergic P2X Receptor Antagonists/pharmacology*
- Rats
- Receptors, Purinergic P2X4/genetics
- Receptors, Purinergic P2X4/metabolism*
- Recombinant Proteins/genetics
- Recombinant Proteins/metabolism
- Species Specificity
- Zebrafish
- PubMed
- 25597706 Full text @ Mol. Pharmacol.
Citation
Ase, A.R., Honson, N.S., Zaghdane, H., Pfeifer, T.A., Seguela, P. (2015) Identification and Characterization of a Selective Allosteric Antagonist of Human P2X4 Receptor Channels. Molecular pharmacology. 87(4):606-16.
Abstract
P2X4 is an ATP-gated nonselective cation channel highly permeable to calcium. There is increasing evidence that this homomeric purinoceptor expressed in several neuronal and immune cell types is involved in chronic pain and inflammation. The current paucity of unambiguous pharmacological tools available to interrogate or modulate P2X4 function led us to pursue the search for selective antagonists. In the high-throughput screen of a compound library, we identified the phenylurea BX430 (MW= 413) with antagonist properties on human P2X4-mediated calcium uptake. Patch-clamp electrophysiology confirmed direct inhibition of P2X4 currents by extracellular BX430 with submicromolar potency (IC50 = 0.54 μM). BX430 is highly selective, having virtually no functional impact on all other P2X subtypes, namely P2X1, P2X2, P2X3, P2X5 and P2X7, at 10 to 100 times its IC50. Unexpected species differences were noticed, as BX430 is a potent antagonist of zebrafish P2X4 but has no effect on rat and mouse P2X4 orthologs. The concentration-response curve for ATP on human P2X4 in presence of BX430 shows insurmountable blockade, indicating a noncompetitive mechanism of action. Using the YO-PRO 1 uptake assay, we observed that BX430 also effectively suppresses ATP-evoked and ivermectin-potentiated membrane permeabilization induced by P2X4 pore dilation. Finally, in single-cell calcium imaging, we validated its selective inhibitory effects on native P2X4 channels at the surface of human THP-1 cells differentiated into macrophages. In summary, this ligand provides a novel molecular probe to assess the specific role of P2X4 in inflammatory and neuropathic conditions, where ATP signaling has been shown to be dysfunctional.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping